2.3 Reagents,
Solutions, and Media
1. Dimethyl sulfoxide (DMSO, Sigma).
2. Ethylene glycol (EG, Fluka).
3. Methylcellulose (Sigma).
4. Ethylene diamine tetraaceticacid tetrasodium salt (EDTA,
Sigma).
5. Reduced glutathione (Sigma).
6. L-Ascorbic acid 2-phosphate magnesium salt (Wako).
7. Primocin (InvivoGen).
8. ROCK Inhibitor (Y-27632, Res Gen).
9. Versene (Gibco).
10. TrypLE Select (Gibco).
11. Recombinant Human Vitronectin (BioLegend).
12. 100Â Glutamax (Gibco).
13. 100Â Non-Essential Amino Acids (NEAA, Gibco).
14. 50Â Essential Amino Acids (EAA, Gibco).
15. RPMI 1640 Vitamins Solution (100Â, Sigma).
16. Dulbecco’s calcium- and magnesium-free phosphate-buffered
saline (DPBSÀ, Gibco).
17. Dulbecco’s phosphate-buffered saline with calcium and magnesium (DPBS+, Gibco).
18. Hanks’ Balanced Salt Solution containing 1.26-mM calcium
and 0.90-mM magnesium without phenol red (HBSS+,
Gibco).
19. Hanks’ Balanced Salt Solution without calcium and magnesium (HBSSÀ, Gibco).
20. DMEM/F-12, HEPES (Gibco).
21. Essential 8 (E8) Flex Medium + Supplement (Gibco).
22. Knock-Out Serum Replacement (Gibco).
23. 10 mM ROCK inhibitor (Y-27632) stock solution: Dissolve
2 mg of Y-27632 in 0.62 mL of PBS to obtain a 10-mM stock
solution, and then aliquot and store the stock solution at
À20
C or À80
C. The stock solution is stable for at least
1 year (see Note 1).
24. 300-mM (100Â) reduced glutathione stock solution: Dissolve
0.47-g reduced glutathione in 5 mL of PBS to prepare a
300-mM stock solution, and then aliquot and store the stock
solution at À20
C or À80
C.
25. 500-mM (100Â) ascorbic acid 2-phosphate stock solution:
Dissolve 0.76-g L-ascorbic acid 2-phosphate (magnesium salt)
in 5 mL of PBS to prepare a 500-mM stock solution, and then
aliquot and store the stock solution at À20
C or À80
C.
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Juliette Seremak and Ali Eroglu